Proteases are used as animal feed additives to improve livestock health and performance, yet few methods for quantifying protease activity in animal feed have been published. Moreover, those methods often involve special equipment, complicated procedures, or specialized knowledge, which make such analyses very difficult to perform at the locations where the results are most useful. In some cases, a qualitative result may be sufficient to determine if the protease was added to the feed at the correct dosage, or if a sufficient amount of enzyme withstood feed production conditions such as pelleting.
Although there are many protease assays known, none of them addresses all of the obstacles inherent in trying to quantify the activity of an exogenously added protease in the context of animal feed. The added protease may adsorb to components of the feed, making it difficult to extract the enzyme. An in-feed method for protease activity must address the presence of specific protease inhibitors present in certain feedstuffs. A highly sensitive assay must be used to detect protease activity in feed due to its typically low inclusion level. Endogenous protein substrate in the feed can mask the presence of protease activity and make it difficult to establish appropriate conditions to accurately quantify the added protease. The capacity of animal feed to act as a good buffer means that careful attention must be paid to adjusting the pH since all enzymes function optimally only within a certain range of pH values. Due to these obstacles to conducting in-feed assays for protease activity, existing methods usually require additional steps, which increase assay complexity and decrease accuracy and precision.
Consequently, painstaking efforts and technical expertise are required to transfer these in-feed methods to the locations where they would be most useful (i.e., feed mills). Alternatively, shipping samples to a central laboratory for analysis incurs additional cost and time, and the feed has often been used before the results are obtained.
Therefore, there is a need for simple, calibrated methods to detect protease activity in feed that can be performed on-site and without the need for sophisticated equipment.